Yeasts from Marine and Estuarine Waters with Different Levels of Pollution in the State of Rio de Janeiro, Brazil

Yeast counts were made at 24 marine and estuarine sites in the vicinity of Rio de Janeiro, Brazil. Mean salinities of estuarine sites ranged from 14.2 to 27.4‰, and mean temperatures ranged from 25 to 28°C. Total coliform counts varied from 80% above 100,000 colony-forming units (CFU)/100 ml at heavily polluted sites to 100% below 100 CFU/100 ml at unpolluted sites. Total yeast counts above 100 CFU/100 ml were typical of heavily and moderately polluted water but atypical of lightly polluted and unpolluted water. Mean total yeast counts were 2,880 CFU/100 ml for heavily polluted sites, 202 CFU/100 ml for moderately polluted sites, and 3 CFU/100 ml for lightly polluted and unpolluted sites. Total yeast counts had a positive response to increased pollution levels, and Candida krusei and phenotypically similar yeasts as a group were prevalent in polluted estuarine water but rare in unpolluted seawater. The 549 strains of yeasts and yeast-like organisms isolated were grouped into 67 species, of which the 21 most prevalent made up 86% of the total yeast population. The prevalent genera in the polluted estuary were Candida, Rhodotorula, Torulopsis, Hanseniaspora, Debaryomyces, and Trichosporon.     

Ecology of Vibrio species, including Vibrio cholerae, in natural waters of Kent, England

The distribution of Vibrio species in water from two sites in Kent was studied between 1978 and 1980. They were counted by a most probable number technique using alkaline peptone water for enrichment followed by plating onto thiosulphate citrate bile salt sucrose agar, or by direct plating of water onto the same agar. In a freshwater stream both upstream and downstream from a human sewage works outfall V. metschnikovii was the predominant Vibrio. Vibrio anguillarum was isolated sporadically. Non-O1 serovars of V. cholerae occurred only twice. At the other site in a ditch containing static, brackish water, non-O1 V. cholerae (highest number 400 cfu/ml) was observed as present only from May to November. Vibrio anguillarum was isolated throughout the sampling period. The presence of non-O1 V. cholerae in both sites was not dependent on the input of human sewage. The hypothesis that non-toxigenic V. cholerae can survive and multiply in water was tested in the ditch by the use of submersible chambers constructed of polycarbonate membranes and Plexiglass. The seasonal incidence of non-O1 V. cholerae in the brackish water site could be explained by the multiplication of the organism when the water temperature exceeded 9°C. It was concluded that strains of V. cholerae that are unable to produce cholera toxin are indigenous to static brackish water environments and the possibility that this applies to toxigenic strains as well should be investigated.     

Impact of industrial waste water effluents on mycoflora of the shore sediments of the 3rd oxidation pond,with reference to biosorption of heavy metals

The third oxidation pond at 10th of Ramadan desert receives a number of industrial waste water effluents contaminated with the heavy metal ions Zn, Cd, Cu and Ni. The species diversity and fungal community structure of seven different sites at the onshore sediments and offshore were studied. Mycological analysis resulted in isolation of 3912 fungal colonies, 11.7% of this count were recovered from the onshore sediment sites (4 sites) whereas 88.3% were from the offshore sites (3 sites), in the desert. Fungal counts and species diversity at the onshore sites tend to increase with increasing distance far from the waste water input. A complete accordance was observed among the total fungal counts and species variabilities with organic matter content at each sampling site. This relationship was reversed in case of heavy metal contents with both counts and diversity. Seventeen fungal species belonging to seven genera were isolated from all sites under study. Aspergillus spp. constituted the majority of the isolates (51.7% of the total isolates), followed by Curvularia, Cephalosporium, and Humicola. Of nine isolated Aspergillus spp., A. humicola was the most dominant (37.4% of the total catch) and appeared at all polluted sites. Therefore, A. humicola was chosen to investigate its potential for heavy metals sorption from the contaminated waste water effluent. Four days old biomass pellets could sorb a large amount of heavy metals according to the following sequence: Zn>Cd>Cu>Ni ions. Agitation significantly increased Zn and Cd sorption, but not Cu and Ni. Heavy metals sorption took place at a wide pH range and particularly increased at alkaline pH (8-9).     

Cadmium exposure of rainbow trout, Salmo gairdneri Richardson: effects on immune functions

Differential leucocyte counts, phagocytosis, humoral antibody response and the in vitro blasto-genetic response to mitogens (lipopolysaccharide and Concanavalin A) and to an antigen ( Vibrio anguillarum ) were studied in rainbow trout exposed to 0,0.7 or 3.6 μg Cd 1⁻¹ for 12 weeks.
Although the fish did not exhibit any clinical or histological changes, cadmium exposure was found to affect two of the immune parameters measured. The cellular response of fish immunized with V. anguillarum to the homologous antigen was significantly lower for splenocytes obtained from fish exposed to cadmium for 9 weeks (3.6 μg Cd 1⁻¹ group) than for splenocytes obtained from non-exposed fish. Conversely, the humoral antibody response to V. anguillarum O-antigen was higher in the 3.6 μg Cd 1⁻¹ group than in the non-exposed group. Protective immunity of fish vaccinated against V. anguillarum was equally as good in the cadmium-exposed group as in the non-exposed group. No cadmium-induced changes in differential leucocyte counts or in the proportions of phagocytic cells were observed.     

Quantitative measurement of endotoxin in rainbow trout (Salmo gairdneri) serum by the chromogenic substrate method

The amount of endotoxin in serum collected from normal rainbow trout ( Salmo gairdneri) and trout inoculated with viable Vibrio anguillarum or lipopolysaccharide (LPS) extracted from bacteria was determined by the chromogenic substrate method. The mean values of endotoxin in four different groups of normal rainbow trout sera ranged from 31.9 to 65.3 pg/ml. When fish were inoculated with viable bacteria (1 × 10⁸), they became septicaemic and a large amount of endotoxin (> 14 ng/ml) was detected in the sera. In fish inoculated with a smaller number of bacteria the amount of endotoxin was several times higher than that of normal fish in spite of failure of bacterial isolation. Although the endotoxin level in serum increased rapidly (> 100 ng/ml) after intraperitoneal inoculation with purified V. anguillarum LPS (540 μg), no fish died during the experiment. The high level of endotoxin in normal rainbow trout and the resistance of trout to endotoxin are in striking contrast to those of mammalian and avian species.     

Isolation and characterization of turbot (Scophtalmus maximus)-associated bacteria with inhibitory effects against Vibrio anguillarum.

More than 400 isolates from the intestine and the external surface of farmed Scophtalmus maximus as well as from fish food and hatchery water were screened for inhibitory effects against the fish pathogen Vibrio anguillarum HI 11345 and seven other fish pathogens. The bacteria with inhibitory effects were then characterized with regard to their sites of colonization, especially the intestinal regions and sites within each region. Of the total number of bacterial isolates from the intestine, 28% were inhibitory against V. anguillarum HI 11345. A marine biochemical assay was used to order the inhibitory strains into different phena. Most inhibitory bacteria were found in the rinse and mucus fractions of the gastrointestinal tract. No correlations among the different phena, site of colonization, and inhibitory effect could be found; however, a biochemical diversity was noted in the strains with an inhibitory effect. Of the isolates with an inhibitory effect against V. anguillarum HI 11345, 60% had an inhibitory effect on five other fish-pathogenic serotypes of V. anguillarum. Inhibitory effects of the isolates were also shown against Aeromonas salmonicida and Aeromonas hydrophila.     

Isolation and characterization of turbot (Scophtalmus maximus)-associated bacteria with inhibitory effects against Vibrio anguillarum.

More than 400 isolates from the intestine and the external surface of farmed Scophtalmus maximus as well as from fish food and hatchery water were screened for inhibitory effects against the fish pathogen Vibrio anguillarum HI 11345 and seven other fish pathogens. The bacteria with inhibitory effects were then characterized with regard to their sites of colonization, especially the intestinal regions and sites within each region. Of the total number of bacterial isolates from the intestine, 28% were inhibitory against V. anguillarum HI 11345. A marine biochemical assay was used to order the inhibitory strains into different phena. Most inhibitory bacteria were found in the rinse and mucus fractions of the gastrointestinal tract. No correlations among the different phena, site of colonization, and inhibitory effect could be found; however, a biochemical diversity was noted in the strains with an inhibitory effect. Of the isolates with an inhibitory effect against V. anguillarum HI 11345, 60% had an inhibitory effect on five other fish-pathogenic serotypes of V. anguillarum. Inhibitory effects of the isolates were also shown against Aeromonas salmonicida and Aeromonas hydrophila.     

Microbial communities in the saturated groundwater environment II: Diversity of bacterial communities in a Pleistocene sand aquifer and their in vitro activities

Bacterial cell numbers obtained from 103 water and sediment samples from a Pleistocene sandy aquifer in the Lower Rhine region (Bocholt, FRG) were determinated on P-agar and by direct count. Below 5 m under the surface, colony-forming unit (cfu) numbers in water samples were less than 100/ml, and in many cases less than 50/ml. In sediment samples, they were 10- to 100-fold higher (10²–10⁴ cfu/g dry wt), but changing markedly between different depths. Direct cell counts yielded numbers two to three orders of magnitude higher.About 2,700 strains of bacteria from 60 samples were isolated randomly and characterized by morphological and physiological properties. Of all the isolates, 71.6% were gram-negative, and 52.2% were gram-negative straight rods. Water communities, with one exception, had low proportions of gram-positive bacteria (<11%), whereas in all but one of the sediment communities percentages of gram-positive isolates were three- to sevenfold higher (35–43%). Water and sediment communities, as well as communities from different sampling sites and communities from different depths of the same sampling site, differed in their qualitative and quantitative morphotype composition and physiological capabilities.The in vitro activities of strains within a single community were quite different, indicating that each community is composed of many diverse bacteria, several having extremely different capabilities. Thus, each community has its own specific activity pattern. Gram-positive bacteria showed on an average lower total activities than did gram-negative bacteria. Grampositive bacteria as well as gram-negative bacteria from sediment had higher values of in vitro activities than the corresponding groups isolated from water. Many water and sediment bacteria preferred the same substrates which were utilized at high rates. However, there were differences in the degradation of the various other substrates present, and each community showed preferences for particular substrates, which they degraded best.The results of cell morphology and physiology studies indicated that all eight characterized communities were very different from one another and very diversely structured.     

Ecology ofVibrio cholerae in the freshwater environs of Calcutta,India

Seasonal incidence ofVibrio cholerae was monitored for a year in a man-made freshwater lake, an open sewage canal, and a pond composed of rainwater accumulations, located in Calcutta.V. cholerae was found in all sites. It exhibited a distinct bimodal seasonal cycle in the lake with a primary peak in August–September and a secondary peak in May–June. Correlation with environmental parameters revealed that temperature and, to a certain extent, pH were the important factors governing the densities ofV. cholerae. In the lake, sediment samples harbored high densities ofV. cholerae immediately after months when peak counts were observed in plankton, suggesting a cycle of cells between sediment and water. At the other sampling areas, no defined seasonality was observed. Instead, high counts ofV. cholerae were observed at these severely polluted sites throughout the study period, including the winter months. All the 15 water samples passed via the ligated loop of rabbits yielded pure cultures ofV. cholerae, indicating that the rabbit intestine selects outV. cholerae from a mixed flora. Uniformly high isolation rates ofV. cholerae were observed from brackish water and freshwater species of export quality prawns.V. cholerae was found to be abundant and was represented by 32 individual Louisiana State University (LSU) serovars, including two new serovars. The 01 serovar could not be isolated from any of the samples examined in this study. It was concluded thatV. cholerae non-01 is common in the freshwater environs of Calcutta.     

Copper as an initiating factor of vibriosis (Vibrio anguillarum) in eel (Anguilla anguilla)

Eels ( Anguilla anguilla ) which were exposed to copper-contaminated fresh water (30–60 ρg Cu/I) died with signs of vibriosis ( Vibrio anguillarum). Eels kept in non-contaminated fresh water (<6ρgCu/I) remained healthy. V. anguillarum was shown to be present in the eels with symptoms of vibriosis. We suggest V. anguillarum is a common inhabitant of eels and copper can change a commensal association between fish and bacterium to one of pathogenicity.     

Microbial colonization of aquifer sediment exposed in a groundwater well in northern Germany

Microbial growth within the water-saturated subsurface environment was investigated by exposing sandy sediments to groundwater for 12 weeks at a depth of 10 or 20 m in a stainless-steel groundwater well. Washing and heating the sediment to 600 degrees C (removal of organic carbon) prior to the exposure did not prevent the natural microbial community from colonizing the sterilized sediment samples. Total cell counts of more than 10(7) or 10(8) per g of dried sediment were obtained. Viable cell counts of 10(5) cells per g on oligotrophic media indicated the presence, within the exposed sediment, of a highly active and multiplying biota. Microscopic analysis of enrichments inoculated with exposed sediment samples revealed a total of 45 different morphotypes, approximately 42% of the microbial community observed in previous studies of this site. The interstitial water running off of the retrieved sediment contained only 17 morphotypes and had up to 6 x 10(5) viable cells per ml.     

Microbial colonization of aquifer sediment exposed in a groundwater well in northern Germany.

Microbial growth within the water-saturated subsurface environment was investigated by exposing sandy sediments to groundwater for 12 weeks at a depth of 10 or 20 m in a stainless-steel groundwater well. Washing and heating the sediment to 600 degrees C (removal of organic carbon) prior to the exposure did not prevent the natural microbial community from colonizing the sterilized sediment samples. Total cell counts of more than 10(7) or 10(8) per g of dried sediment were obtained. Viable cell counts of 10(5) cells per g on oligotrophic media indicated the presence, within the exposed sediment, of a highly active and multiplying biota. Microscopic analysis of enrichments inoculated with exposed sediment samples revealed a total of 45 different morphotypes, approximately 42% of the microbial community observed in previous studies of this site. The interstitial water running off of the retrieved sediment contained only 17 morphotypes and had up to 6 x 10(5) viable cells per ml.     

Inhibition of vibrio anguillarum by Pseudomonas fluorescens AH2, a possible probiotic treatment of fish

To study the possible use of probiotics in fish farming, we evaluated the in vitro and in vivo antagonism of antibacterial strain Pseudomonas fluorescens strain AH2 against the fish-pathogenic bacterium Vibrio anguillarum. As iron is important in virulence and bacterial interactions, the effect of P. fluorescens AH2 was studied under iron-rich and iron-limited conditions. Sterile-filtered culture supernatants from iron-limited P. fluorescens AH2 inhibited the growth of V. anguillarum, whereas sterile-filtered supernatants from iron-replete cultures of P. fluorescens AH2 did not. P. fluorescens AH2 inhibited the growth of V. anguillarum during coculture, independently of the iron concentration, when the initial count of the antagonist was 100 to 1, 000 times greater that of the fish pathogen. These in vitro results were successfully repeated in vivo. A probiotic effect in vivo was tested by exposing rainbow trout (Oncorynchus mykiss Walbaum) to P. fluorescens AH2 at a density of 10(5) CFU/ml for 5 days before a challenge with V. anguillarum at 10(4) to 10(5) CFU/ml for 1 h. Some fish were also exposed to P. fluorescens AH2 at 10(7) CFU/ml during the 1-h infection. The combined probiotic treatment resulted in a 46% reduction of calculated accumulated mortality; accumulated mortality was 25% after 7 days at 12 degrees C in the probiotic-treated fish, whereas mortality was 47% in fish not treated with the probiont.     

Identification of Vibrio anguillarum in fish by using partial 16S rRNA sequences and a specific 16S rRNA oligonucleotide probe.

16S rRNA from seven different Vibrio anguillarum strains was partially sequenced and compared. From this sequence information we could design a 25-base-long oligonucleotide and use it as a specific probe for identification of V. anguillarum. This was determined by RNA-DNA colony hybridization and slot-blot hybridization. Strong, specific hybridization to the probe was observed for all V. anguillarum strains tested. Furthermore, no cross-hybridization could be seen against five other bacterial species. The detection limit was 5 x 10(3) bacteria per ml. It was even possible to detect V. anguillarum, by slot-blot hybridization, directly in a homogenized kidney from a fish that had died of vibriosis. The partial sequence information revealed small but significant differences between strains of the same species. These sequence differences are sufficiently significant to allow serotyping on the RNA level. Comparing strains of different serotypes revealed a 10-base and an 11-base difference in V. anguillarum serotypes O8 and O9, respectively, in a 122-base partial sequence.     

Identification of Vibrio anguillarum in fish by using partial 16S rRNA sequences and a specific 16S rRNA oligonucleotide probe

16S rRNA from seven different Vibrio anguillarum strains was partially sequenced and compared. From this sequence information we could design a 25-base-long oligonucleotide and use it as a specific probe for identification of V. anguillarum. This was determined by RNA-DNA colony hybridization and slot-blot hybridization. Strong, specific hybridization to the probe was observed for all V. anguillarum strains tested. Furthermore, no cross-hybridization could be seen against five other bacterial species. The detection limit was 5 x 10(3) bacteria per ml. It was even possible to detect V. anguillarum, by slot-blot hybridization, directly in a homogenized kidney from a fish that had died of vibriosis. The partial sequence information revealed small but significant differences between strains of the same species. These sequence differences are sufficiently significant to allow serotyping on the RNA level. Comparing strains of different serotypes revealed a 10-base and an 11-base difference in V. anguillarum serotypes O8 and O9, respectively, in a 122-base partial sequence.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum.

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Mutagenicity of sediment and biomarkers of oxidative stress in fish from aquatic environments under the influence of tanneries

The mutagenicity of interstitial water and organic extracts from the sediments in the Cadeia and Feitoria Rivers, RS, Brazil, were evaluated by Salmonella microsuspension bioassay using TA97a, TA98, TA100 and TA102 strains, in the absence and presence of S9 mix. At the contaminated site, the mutagenic responses for interstitial water, suggested the presence of frameshift and base pair substitution mutagens, including oxidative substances. Organic extracts presented direct or indicative mutagenesis to the TA97a, TA98 and TA100 strains. In general, an exogenous metabolic systems decreased the mutagenicity of the samples. High concentrations of total chromium found in the sediment and interstitial water as well as total mercury in the sediment of the contaminated site, when compared to the control area, may help explain the mutagenic results. The livers of Gymnogeophagus gymnogenys collected in this impacted area, compared to a non-polluted site, were analyzed for oxidative stress parameters. Compared to the controls, there was a significant increase in the activity of superoxide dismutase (SOD) at levels of substances reactive to thiobarbituric acid (TBARS), and in the chemiluminescence of hepatic cells in fish in the polluted area. The concentration of cytochromes P450 and b5 decreased drastically in the fish at the polluted site, while the catalase activity did not change. It was possible to correlate the biological changes in the fish with the presence of mutagenic compounds in sediment and interstitial water in this area.     

Sediment bacterial indicators in an urban shellfishing subestuary of the lower Chesapeake Bay.

The objectives of this study were to document the spatial and temporal distributions and compositions of bacteria in the sediments and overlying waters of an important urban shellfishing area in the lower Chesapeake Bay region, the Lynnhaven Estuary. Marked fluctuations were observed in the date of many of the physicochemical parameters and the indicator bacteria. The higher-salinity water and coarser sediment of the inlet site showed lower overall bacterial densities than did the headwater sites, where freshwater runoff and decreased tidal action were characteristic. Densities of benthic indicator bacteria, when expressed on a volumetric basis, were significantly greater than counts in the overlying waters. These counts were indicative of a fecally polluted system and were well above the safe maximum limits for shellfish-growing waters. Significantly fewer total and fecal bacteria were observed in both the water and the sediment during the warm months of May, July, and August. The primary sources of the Lynnhaven's bacterial pollution appeared to be typical of urban and agricultural runoff, although failure of septic tank systems was suspected as a problem in the Lynnhaven's western branch. These results illustrated that sediments in shellfishing areas could serve as a reservoir for high densities of indicator bacteria and that, potentially, pathogens could pose a health hazard.     

Quantitative measurement of endotoxin in rainbow trout (Salmo gairdneri) serum by the chromogenic substrate method

The amount of endotoxin in serum collected from normal rainbow trout (Salmo gairdneri) and trout inoculated with viable Vibrio anguillarum or lipopolysaccharide (LPS) extracted from bacteria was determined by the chromogenic substrate method. The mean values of endotoxin in four different groups of normal rainbow trout sera ranged from 31.9 to 65.3 pg/ml. When fish were inoculated with viable bacteria (1 x 10(8], they became septicaemic and a large amount of endotoxin ng/ml) was detected in the sera. In fish inoculated with a smaller number of bacteria the amount of endotoxin was several times higher than that of normal fish in spite of failure of bacterial isolation. Although the endotoxin level in serum increased rapidly (greater than 100 ng/ml) after intraperitoneal inoculation with purified V. anguillarum LPS (540 micrograms), no fish died during the experiment. The high level of endotoxin in normal rainbow trout and the resistance of trout to endotoxin are in striking contrast to those of mammalian and avian species.